Protective effects of the postbiotic deriving from cow’s milk fermentation with L. paracasei CBA L74 against Rotavirus infection in human enterocytes

Rotavirus (RV) is the leading cause of acute gastroenteritis-associated mortality in early childhood. Emerging clinical evidence suggest the efficacy of the postbiotic approach based on cow’s milk fermentation with the probiotic Lacticaseibacillus paracasei CBAL74 (FM-CBAL74) in preventing pediatric acute gastroenteritis, but the mechanisms of action are still poorly characterized. We evaluated the protective action of FM-CBAL74 in an in vitro model of RV infection in human enterocytes. The number of infected cells together with the relevant aspects of RV infection were assessed: epithelial barrier damage (tight-junction proteins and transepithelial electrical resistance evaluation), and inflammation (reactive oxygen species, pro-inflammatory cytokines IL-6, IL-8 and TNF-α, and mitogen-activated protein kinase pathway activation). Pre-incubation with FM-CBA L74 resulted in an inhibition of epithelial barrier damage and inflammation mediated by mitogen-activated protein kinase pathway activation induced by RV infection. Modulating several protective mechanisms, the postbiotic FM-CBAL74 exerted a preventive action against RV infection. This approach could be a disrupting nutritional strategy against one of the most common killers for the pediatric age.


Results
Cellular damage. The RV infectivity is commonly demonstrated by the immunofluorescence staining of viral capsid protein VP6 in human enterocytes 17,18 . The RV infection of Caco-2 cells was confirmed by an increase of VP6 protein quantification and mRNA levels (p < 0.001) (Fig. 1, panel A). The co-incubation of RV with FM-CBAL74 for 6 h before infection or the pre-incubation of RV with FM-CBAL74 for 48 h before infection were unable to limit the number of infected enterocytes (Fig. 1, panel A).
RV infection induces alterations of the cytoskeleton through ERK pathway activation via phosphorylating actin cross-linking/bundling proteins 19 . This mechanism induces a rearrangement of F-actin filaments 20 . As showed in Fig. 1, panel B, non-infected cells showed a regular distribution of cytoskeleton actin filaments. In contrast, RV-infected enterocytes showed a marked alteration of cytoskeleton structure with a disorganization of F-actin filaments. Pre-incubation with FM-CBAL74, but not with non-fermented cow's milk (NFM), protected the cells against RV-induced rearrangements of F-actin filaments (Fig. 1, panel B).
RV infection induces apoptosis in human enterocytes 21,22 . An increase in necrotic cells (positive only for propidium, PI) and late apoptotic cells (positive for both PI and Annexin V) confirmed the pro-apoptotic effect induced by RV infection (p < 0.05) (Fig. 1, panel C). Pre-incubation with FM-CBAL74, but not with NFM, prevented these effects (p < 0.05) (Fig. 1, panel C).
These results suggested that, while FM-CBAL74 is unable to prevent RV infection in human enterocytes, it can limit the subsequent cytoskeleton alterations and apoptosis.
MAP kinases pathway activation. The activation of the extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK), has been described in RNA viruses' infection 23 . The phosphorylation status of these kinases was investigated. Pre-treatment with FM-CBAL74 prevented the RV-induced phosphorylation ratio increase of ERK and JNK (p < 0.001) (Fig. 2, panels A,B).
These results provided evidence on ERK/JNK pathway involvement in the protective action of FM-CBAL74 against RV infection.
Intestinal permeability. To investigate the protective action of FM-CBAL74 against gut barrier alteration induced by RV infection, we evaluated the transepithelial electric resistance (TEER), the expression of the tight junction (TJ) proteins, occludin and zonula occludens -1 (ZO-1), and of the cell adhesion molecule E-cadherin in Caco-2 cells monolayer.
RV infection determined a significant decrease of transepithelial resistance (TEER) in human enterocytes (p < 0.05). This event was associated with an alteration of TJ proteins structure, as demonstrated by redistribution of occludin, ZO-1 and E-cadherin (Fig. 3). The pre-treatment with FM-CBAL74 prevented RV-induced TEER decrease (p < 0.05) (Fig. 3, panel A). The redistribution of occludin and ZO-1 has been associated with TJ proteins alteration and barrier dysfunction in gut epithelium 24 . RV infection in Caco-2 cells caused an alteration of TJ proteins, as demonstrated by occludin and ZO-1 redistribution (Fig. 3, panels B,C) and by reduction of their expression (p < 0.05) (Fig. 3, panels B,C). Pre-treatment with FM-CBAL74, but not with NFM, protected Caco-2 cells from occludin and ZO-1 redistribution (Fig. 3, panel B,C) and enhanced their expression (p < 0.05) ( Fig. 3 panels B,C), suggesting a protective effect against gut barrier dysfunction. Furthermore, we found that E-cadherin protein appeared significantly reduced in RV-infected cells (p < 0.001) (Fig. 3, panel D). Again, pretreatment with FM-CBAL74, but not with NFM, significantly prevented the reduction of E-cadherin expression caused by RV infection (p < 0.05) (Fig. 3, panel D).
These results showed that FM-CBAL74 was able to prevent RV induced alteration of gut barrier integrity. In Supplementary Information 3, we provide a 3D video reconstruction from Z-stack acquisition of Occludin.
Inflammatory response. Oxidative stress and inflammation are closely related pathophysiological events in infectious diseases 25 . To further investigate the protective action of FM-CBAL74 on inflammatory response induced by RV infection, we investigated oxidative stress (ROS production) and pro-inflammatory cytokines (IL-6, IL-8 and TNF-α) response in human enterocytes.
As shown in Fig. 4, panel A, RV significantly increased ROS production. The pretreatment with FM-CBAL74, but not with NFM, inhibited the RV-induced ROS increase (p < 0.05) (Fig. 4, panel A). In parallel, FM-CBAL74, but not NFM, inhibited IL-6, IL-8 and TNF-α production induced by RV infection in human enterocytes (p < 0.05) (Fig. 4,  www.nature.com/scientificreports/ Altogether these data suggested that FM-CBAL74 was able to inhibit ROS production and IL-8, IL-6 and TNF-α cytokines release induced by RV infection in human enterocytes.

Discussion
Emerging evidence suggest the potential of the postbiotic approach for the prevention of pediatric infectious diseases [6][7][8][9][10][11][12][13][14][15][16]26,27 . Unfortunately, discrepancies in clinical results evaluating different postbiotic products, and the poor definition of the mechanisms of action elicited against specific pathogens are blunting the wide use of this approach in clinical practice.
Rotavirus is the most common agent of AGE in the pediatric age 2,3 . Our study aimed to explore the anti-RV effect of a specific postbiotic deriving from the fermentation of cow's milk with the probiotic L. paracasei CBAL74 with a demonstrated clinical efficacy against pediatric AGE 11,12 . We found that this postbiotic positively modulates a range of non-immune and immune defense mechanisms against RV infection in human enterocytes.
Even though this postbiotic was unable to significantly reduce the number of infected cells, a protective action against gut barrier damage, characterized by cytoskeleton alterations and apoptosis, was observed. The effect involved the modulation of a pivotal regulator of stress-induced cell damage, the ERK/JNK kinase pathway [28][29][30][31][32] . ERK/JNK pathway is activated by different viruses, including influenza A, herpes simplex virus 1, hepatitis C www.nature.com/scientificreports/ virus, and RV 23,32-35 . Components of RV outer capsid proteins, such as VP4-VP8 and VP4-VP5 domains, VP7, and the non-structural proteins NSP1 activate PI3K/Akt and ERK/JNK pathways 23,36 . According to previous evidence 37,38 , our data demonstrated that RV infection stimulates ROS production in human enterocytes. The increased ROS production induces the release of pro-inflammatory cytokines (IL-8, IL-6, TNF-α) through the activation of MAP kinase pathway 25,[39][40][41][42] . We demonstrated that FM-CBAL74 was able to prevent RV-induced ROS, IL-8, IL-6 and TNF-α production, suggesting an inhibition of the inflammatory "cytokine storm" which in turn is responsible for the severity of signs and symptoms commonly observed in children infected by this microorganism 2 .
A major strength of our study is the evaluation of all crucial steps of RV infection in a well validated experimental model [43][44][45][46] . The major limitation resides in the lack of evidence on which specific FM-CBAL74 component could be responsible for the protective effects. Several components of this postbiotic could be involved, including lipoteichoic acid, peptidoglycans, bacteriocins, nucleotides and peptides. It has been demonstrated that peptides deriving from fermentation of cow's milk proteins could act as modulators of non-immune and immune gastrointestinal defense mechanisms [47][48][49] . We have previously demonstrated that this postbiotic could exert a positive modulation of gut microbiome in the pediatric age associated with increased production of secretory IgA (sIgA) and butyrate [14][15][16] . Considering the relevant role exerted by these molecules in protecting against infections and in modulating gut barrier integrity and inflammation, it is possible to hypothesized that the protective actions reported in this study could be further reinforced by the modulation of sIgA and gut microbiome 10 . Future studies on the efficacy and mechanisms of action of FM-CBAL74 using more complex systems, such as human biopsies and/or organoids exposed to different gastrointestinal pathogens, are advocated to further explore the potential of this approach. Another limitation is related to the fact that we explored the protective effects of FM-CBAL74 against only the main agent of pediatric AGE. Similar protective action against Salmonella typhimurium has been reported by others 13 . Future studies are advocated to better elucidate the protective effect elicited by this postbiotic against other microorganisms responsible for pediatric AGE.
In conclusion, we provided evidence on the protective action elicited by FM-CBAL74 against the most common agent of pediatric AGE. A range of intracellular mechanisms has been highlighted. These mechanisms could act in parallel with other beneficial actions on gut microbiome structure and function, and on innate and adaptive immunity that has been already demonstrated in children receiving this postbiotic [11][12][13][14][15][16] . Altogether, these data www.nature.com/scientificreports/ could pave the way to innovative nutritional strategies against one of the most common killers for the pediatric age, responsible for > 600,000 deaths yearly worldwide 3 .

Rotavirus activation and infection protocol.
Rotavirus strain SA11 activation was performed as previously described 43

Apoptosis (annexin V assay) by FACS analysis.
To analyze cell apoptosis rate, 2.5 × 10 5 undifferentiated Caco-2 cells were plate in 6-well plates and Annexin V Apopstosis Detection Kit APC was used (eBioscience, San Diego, CA, USA) according to the manufacturer's protocol, as previously described 17  Reactive oxygen species production. Reactive oxygen species (ROS) production was measured by 7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich) spectrofluorometry on differentiated Caco-2 cells, as previously described 43 . Briefly, after stimulation, DCFH-DA (20 µM) was added for 30 min at 37 °C in the dark. After twice washes in PBS, intracellular ROS production was measured in a fluorometer (SFM 25, Kontron Instruments; Japan). As a positive control, hydrogen peroxide (H 2 O 2 ) (Sigma-Aldrich) was used at concentrations of 10 mM for 15, 30 and 60 min.
Quantitative real-time PCR. Total RNA was isolated from cells with TRizol reagent (Sigma-Aldrich) and quantified using a NanoDrop Spectrophotometer and purity was verified by A260/280 and A260/230 absorbance ratios. The integrity of the RNA was checked using gel electrophoresis. RNA (500 ng) was reverse transcribed in cDNA with a High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems; Vilnius, Lithuania) according to the manufacturer's instructions. Complementary DNA (cDNA) was stored at − 80 °C until use. Quantitative real-time PCR (qRT-PCR) analysis was performed using Taqman Gene Expression Master Mix (Applied Biosystems, Grand Island, NY, USA) to evaluate the effect of intestinal exposure to milk products and Rotavirus SA11 on the gene expression of TJ occludin and ZO-1 (Hs00170162_m1 and Hs01551871_m1, respectively). The TaqMan probes for these genes were inventoried and tested by Applied Biosystems manufacturing facility (QC). RV-VP6 expression was evaluated using a SYBR green Master Mix (Applied Biosystems, Grand Island, NY, USA). The primers used were: VP6 F 5′-GCA CAG CCA TTC GAA CAT CATGC-3′; VP6 R 5′-TGC ATC GGC GAG TAC AGA CTC-3′. Amplification conditions were initial steps at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min in a Light Cycler 7900HT (Applied Biosystems). The expression of each gene was normalized to that of Glyceraldeide-3-Phosphate Dehydrogenase (TaqMan assay: GAPDH; Hs02786624_g1, primers for SYBR Green assay: GAPDH F 5′-AAT CCC ATC ACC ATC TTC CAG-3′; GAPDH R 5′-AAT GAG CCC CAG CCTTC-3′) to normalize a relative transcript level. Relative gene expression was calculated by the 2 −ΔΔCT method: (ΔΔCT = ΔCT sample − ΔCT control ). Each sample was analyzed in triplicate.
Analysis of pro-inflammatory cytokines production. The concentrations of IL-8, IL-6 and TNF-α were analyzed in cell supernatants collected after treatment and stored at − 80 °C. The three cytokines production was measured by ELISA using commercially available kits (Abcam, Cambridge, USA) according to the manufacturer's instructions, and results expressed in pg/mL. The detection limits of IL-8, IL-6 and TNF-α were 1.8 pg/ mL, 2 pg/mL and 30 pg/mL, respectively. Western blot analysis. Western blotting analysis was carried out following proteins cell extraction by RIPA buffer (50 mM Tris-Hcl, pH 7.6, 150 mM NaCl, 1 mM MgCl 2 , 1% NP-40) supplemented with a protease and phosphatase inhibitor cocktail. Protein concentrations were estimated using BioRad protein assay dye reagent and BSA (PanReac AppliChem) as standard. Proteins (30 µg) were separated by SDS-Polyacrylamide gel electrophoresis and subsequently transferred onto Polyvinylidene fluoride (PVDF) membranes (Immobilon R -Transfer  (Left (B,C)). Occludin and ZO-1 were visualized with antioccludin and Alexa Fluor-488-conjugated secondary antibody (green) and with anti-ZO-1 and Alexa Fluor-488 -conjugated secondary antibody (green) and nuclei were stained with DAPI (blue). Cells were observed through confocal microscope in the zy-plane. RV infection elicited a redistribution of occludin and ZO-1 proteins in Caco-2 cells and reduced their expression (Right (B,C)). Pretreatment of RV-infected cells with FM-CBAL74 (FM-CBAL74 + RV) prevented the redistribution of occludin and ZO-1 proteins (Left (B,C)) and their mRNA reduction in Caco-2 cells monolayer (Right (B,C)  Immunofluorescence and confocal microscopy. For actin cytoskeleton detection, 2.5 × 10 5 undifferentiated Caco-2 cells were washed and fixed with 4% paraformaldehyde (PFA) (Carlo Erba Reagents) for 10 min at room temperature. Autofluorescence due to free aldehyde groups from PFA treatment were blocked with 50 mM Ammonium Chloride (Sigma-Aldrich) in PBS for 10 min at room temperature. Cover slips were washed twice with PBS, then cells were permeabilized with Triton X-100 (PanReac AppliChem) in PBS for 10 min. After washing, the cells were blocked for 1 h using 1% BSA in PBS/Tween 20 and then incubated for 1 h at room temperature with phalloidin-TRITC (Sigma-Aldrich). At the end, the cells were washed with PBS and mounted with antifading Mowiol. Glass slides were allowed to cure overnight, in the dark.